Elastin is a protein capable of spontaneously recoiling after being stretched. Cross-linked elastin is the major structural component of elastic fibers, which confers tissue elasticity. A proteinase may be named an elastase if it possesses the ability to solubilize mature, cross-linked elastin (Bieth, J G “Elastases: catalytic and biological properties,” at pp. 217-320 (Mecham Edition, Regulation of Matrix Accumulation, New York, Academic Press, 1986). Several published patent applications (WO 2001/21574; WO 2004/073504; and WO 2006/036804) indicate that elastase, alone and in combination with other agents, is beneficial in the treatment of diseases of biological conduits, including biological conduits which are experiencing, or susceptible to experiencing, obstruction and vasospasm. For elastase therapy of human subjects, the use of a human elastase is desirable to reduce the risk of immune reaction to a non-human elastase.
To this date, however, there is no known commercially viable means of producing biologically active elastase in sufficiently pure form and in sufficient quantities for clinical applications. Because elastases are powerful proteases that can hydrolyze numerous proteins other than elastin, the proteolytic activity of elastase poses potential obstacles for its recombinant production. For example, the activity of mature elastase can damage the host cell which is expressing it, degrade itself, or degrade agents used to assist in the production or characterization of the elastase.
Elastases are often expressed as preproproteins, containing a signal peptide, an activation peptide, and a mature, active portion. Cleavage of the signal sequence upon secretion yields a proprotein that can have little or no enzymatic activity, and whose amino acid sequence contains the amino acid sequence of an activation peptide and a mature protein. Generally, for recombinant expression, an inactive precursor may be expressed instead of the mature active enzyme to circumvent damage to the cell that expresses it. For example, U.S. Pat. No. 5,212,068 describes the cloning of human pancreatic elastase cDNAs (referred to therein as elastase “IIA,” “IIB,” “IIIA” and “IIIB”). The various elastases were expressed as full-length cDNAs, including the native signal sequences, in mammalian COS-1 cells. In addition, engineered versions of the elastases, containing a B. subtilis signal sequence and a β-galactosidase signal sequence, were also expressed in B. subtilis and E. coli, respectively. U.S. Pat. No. 5,212,068 also suggests expressing elastases in S. cerevisiae. Generally, working examples of elastase expression in U.S. Pat. No. 5,212,068 show low activity of the recovered elastase or require an activation step involving treatment with trypsin, to generate the active elastase. In addition, the elastases were largely present in inclusion bodies when expressed in E. coli, and only small portions of the expressed elastase were soluble and active. None of the elastase preparations described in U.S. Pat. No. 5,212,068 was purified to pharmaceutical grade.
Thus, there is a need in the art for recombinant manufacturing methods that allow the generation of therapeutic amounts of biologically active pharmaceutical grade elastases, and preferably avoid a trypsin activation step that is costly for large-scale preparation and can result in trypsin contamination of the final product. Administration of an elastase containing trypsin to a patient could result in activation of the protease-activated receptors 1 and 2, which may reduce some of the beneficial effects of elastase treatment.
Citation or identification of any reference in Section 2 or in any other section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.